Inhibition of ornithine aminotransferase for the treatment of proliferative disorders

ABSTRACT

The invention relates to inhibition of ornithine aminotransferase (OAT) for suppression of tumor cells proliferation. More particularly, the invention relates to methods of treatment of proliferative disorders by the selective inhibition of OAT, and further provides the use of OAT inhibitors, specifically, 5-amino-1,3-hexadienyl-carboxylic acid (Gabaculine), and Gabaculine analogue 8, for compositions and methods for the treatment of proliferative disorders such as hepatocellular carcinoma. The invention further provides methods and kits for the diagnosis of a pathologic proliferative disorder in a mammalian subject, based on determining the level of OAT expressed in a biological sample obtained from a subject.

FIELD OF THE INVENTION

The invention relates to inhibition of ornithine aminotransferase (OAT)for suppression of tumor cells proliferation. More particularly, theinvention relates to methods of treatment of proliferative disorders byinhibition of OAT, and further provides the use of OAT inhibitors, forcompositions and methods for the treatment of proliferative disorderssuch as hepatocellular carcinoma.

BACKGROUND OF THE INVENTION

All publications mentioned throughout this application are fullyincorporated herein by reference, including all references citedtherein.

Cancer of all forms is one of the major causes of morbidity throughoutthe world. Research in the area of cancer chemotherapy has produced avariety of antitumor agents which have differing degrees of efficacy. Avariety of cancer therapeutic agents are known, for example, alkylatingagents, antimetabolites, alkaloids and carcinostatic antibiotics.Standard clinically used agents include adriamycin, actinomycin D,methotrexate, 5-fluorouracil, cis platinum, vincristine and vinblastine.However, the presently available antitumor agents are known to havevarious disadvantages such as toxicity to healthy cells and resistanceof certain tumor types.

Hepatocellular carcinoma (HCC) is one of the major malignant diseases inthe world today, the greatest incidence being in Japan, China, otherparts of the Asia and sub-Saharan Africa. Recent evidence indicates thatthe incidence of hepatocellular carcinoma in Europe and North America isincreasing. The disease is estimated to be responsible for, or involvedin, up to approximately 1,250,000 deaths a year, and as such it isnumerically one of the world's major malignant diseases.

The prognosis of HCC is poor, with the world-wide frequency rate almostequalling the mortality rate. After diagnosis, the median survival timeis less than four months. Long-term survival, defined as survival longerthan one year after the diagnosis, is seen only occasionally. Most HCCpatients succumb to either the complications of liver failure with orwithout massive bleeding, or to the general effects of a large tumorburden, with cachexia, malnutrition, infection and sepsis. Thoughdistant metastases occur (up to 90% of patients have metastatic tumorsat the time of death), hepatic disease most often limits survival.

Current therapies available to the clinician are on the wholeineffective as a cure for this disease. For patients with advanced HCCwho are not candidates for surgical resection, liver transplantation,localized tumor ablation or systemic chemotherapy remains the mainstayof therapy. Unfortunately, HCC is a relatively chemotherapy-resistantproliferative disorder; therefore, outcomes using this mode of treatmentare unsatisfactory. Resistance to chemotherapy may be caused by theuniversal expression of the multidrug resistance gene protein on thesurface of the malignant cells, leading to active efflux ofchemotherapeutic agents. Chemotherapy is usually not well tolerated andseems to be less efficacious in patients with HCC with underlyinghepatic dysfunction. Younger patients with well-compensated cirrhosisdue to chronic hepatitis B or C infections have better outcome withchemotherapy than older patients with alcoholic cirrhosis and othercomorbid diseases.

The most active single agent drugs tested have been doxorubicin,cisplatin, and fluorouracil. Response rates are about 10%, and treatmentshows no clear impact on overall survival. More recently, gemcitabineand capecitabine have been evaluated in clinical trials, but responserates have been low and short term.

A variety of combination chemotherapy regimens have also been studied.Recently, cisplatin-based combination regimens have shown improvedresponse rates around 20%, but to date, no survival advantage ascompared to supportive care alone has been shown. No difference seems toexist in response rates between 2 or 3-drug regimens. Moreover, some ofthese combination regimens cause considerable toxicity.

Chemoimmunotherapy uses a combination of chemotherapy andimmunomodulatory agents, such as interferon-alpha, to try to achievebetter tumor response rates. Antiangiogenesis agents (i.e.,bevacizumab), which work by disrupting the formation of blood vesselsthat feed tumors, are a new class of drugs that may prove to be ofbenefit in the treatment of HCC. The highly vascular nature of HCCtumors makes therapy with an antiangiogenesis agent a promising andexciting new option. Further evaluation of these drugs in the setting ofclinical trials is needed to determine their efficacy.

Thus, there is a clear need for novel therapeutic approaches forspecifically affecting cancerous cells, and especially, HCC. The presentinvention showed for the first time that ornithine aminotransferase isoverexpressed in malignant proliferative tissue. The inventors furthershowed that inhibition of the enzyme catalytic activity using twodifferent inhibitors, markedly decreased tumor growth, and therefore maybe used as a specific target in the treatment of proliferativedisorders, and particularly of HCC.

Ornithine aminotransferase (OAT) is a mitochondrial matrix enzyme thatcatalyzes a reversible reaction of interconversion between ornithine andalpha ketoglutarate to delta-1-pyrroline-5-carboxylate and glutamate.The enzyme is expressed in many tissues, including liver, kidney, smallintestine, brain and eye. The enzymes from liver and kidney differsignificantly in their regulation, and were believed to be two distinctenzymes. However, DNA sequencing proved that the two enzymes are encodedby a single gene.

As indicated above, glutamate is the product of the reaction catalyzedby OAT. This product can be used as a substrate by glutamine synthetaseto synthesize glutamine, which is critical for the growth ofproliferative cells, supporting protein and nucleotide synthesis andproviding a major source of energy. Therefore an increased activity ofOAT could make a tumor cell independent of any glutamine supply andconfer a growth advantage to the cell. Thus, without being bound by anytheory, it may be hypothesized that reducing the level of tissueglutamine concentrations by inactivation of OAT may lead to inhibitionin cell proliferation and tumor growth.

Furthermore, it should be noted that ornithine is a substrate in theurea cycle. The urea cycle is effective in incorporating ammonium ionsinto urea in order to be eliminated from the body. The present inventionis based on inhibition or inactivation of ornithine aminotransferase(OAT). Therefore, alternatively or additionally and without being boundby any theory, it may be postulated that by enhancing the level oftissue ornithine concentrations due to inactivation of OAT over anextended period of time, urea formation in the liver and presumably insome other tissues would be a consequence thereof, thereby loweringblood and cerebrospinal fluid ammonia concentrations. These compoundswere implicated in numerous well known human illnesses associated withelevated blood and cerebrospinal fluid ammonia concentrations, amongwhich, for example, are liver cirrhosis, fulminant hepatic failure andurinary tract/bladder infections. These, and particularly cirrhosis, maytherefore lead to HCC.

Thus, the present invention comprises the new use of OAT inhibitors, forthe treatment of proliferative disorders.

It is thus one object of the invention to provide a method for thetreatment of proliferative disorders, and particularly HCC, which isbased on inhibiting the expression or the activity of ornithineaminotransferase (OAT).

Another object of the invention is the use of ornithine aminotransferaseinhibitors, and particularly of Gabaculine and Gabaculine analogue 8,for the preparation of pharmaceutical composition for the treatment ofmalignant proliferative disorders.

Another object of the invention is to provide for a diagnostic methodfor detection of proliferative disorders, particularly of HCC, bydetecting increase in the expression of ornithine aminotransferase.

These and other objects of the invention will become apparent as thedescription proceeds.

SUMMARY OF THE INVENTION

In a first aspect the present invention relates to a method for thetreatment of a pathologic proliferative disorder in a subject in needthereof. The method of the invention is based on selectively inhibitingornithine aminotransferase (OAT) in the treated subject.

According to one embodiment, the inhibition of ornithineaminotransferase (OAT) may be achieved by inhibiting the expression orby inhibiting the enzymatic activity of this enzyme.

In a specifically preferred embodiment, inhibiting the enzymaticactivity of ornithine aminotransferase (OAT) may be performed byadministering to the treated subject an inhibitory effective amount ofOAT inhibitor selected from the group consisting of5-amino-1,3-hexadienyl-carboxylic acid (Gabaculine), Gabaculine analogue8 (LHJ-II-79), 5-fluoromethylornithine (6-fluoro-2,5-diaminohexanoicacid, 5-FMOrn) and (S)-2-amino-4-amino-oxybutyric acid (Canaline). Morepreferably, the inhibitor may be 5-amino-1,3-hexadienyl-carboxylic acid(Gabaculine). According to another preferred embodiment, the inhibitormay be Gabaculine analogue 8 (LHJ-II-79).

In a second aspect, the invention relates to a pharmaceuticalcomposition for the treatment of a pathologic proliferative disorder.The composition of the invention comprises as an active ingredient anornithine aminotransferase (OAT) inhibitor, that may be capable ofinhibiting the expression or the enzymatic activity of said OAT.

In a further aspect, the invention provides for the use of an ornithineaminotransferase (OAT) inhibitor for the preparation of a pharmaceuticalcomposition for the treatment of a pathologic proliferative disorder ina subject in need thereof.

In a fourth aspect, the invention relates to a diagnostic method fordiagnosis of a pathologic proliferative disorder. The diagnostic methodof the invention comprises the steps of: (a) determining the level ofOAT expressed in a biological sample obtained from said subject; (b)determining the level of one or more control genes expressed in saidbiological sample obtained from said subject; and (c) comparing thelevel of expression of OAT in said sample according to step a) and thelevel of expression of each of said one or more control genes in saidsample according to step b) with the level of OAT and the level of oneor more control reference genes in a control sample; wherein detectingdifferential expression of OAT in the comparison of step c) isindicative of that said subject is suffering of said proliferativedisorder.

The invention further provides kits for the diagnosis of pathologicproliferative disorders.

These and more aspects of the invention will become apparent in hand ofthe following examples.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect the present invention relates to a method for thetreatment of a pathologic proliferative disorder in a subject in needthereof. The method of the invention is based on the step of selectivelyinhibiting ornithine aminotransferase (OAT) in the treated subject.

According to one embodiment, the inhibition of ornithineaminotransferase (OAT) may be achieved by inhibiting the expression orby inhibiting the enzymatic activity of this enzyme.

In a specifically preferred embodiment, inhibiting the enzymaticactivity of ornithine aminotransferase (OAT) may be performed byadministering to the treated subject an inhibitory effective amount ofOAT inhibitor selected from the group consisting of5-amino-1,3-hexadienyl-carboxylic acid (Gabaculine), Gabaculine analogue8 (LHJ-II-79), 5-fluoromethylornithine (6-fluoro-2,5-diaminohexanoicacid, 5-FMOrn) and (S)-2-amino-4-amino-oxybutyric acid (Canaline. Morepreferably, the inhibitor may be 5-amino-1,3-hexadienyl-carboxylic acid(Gabaculine). It should be noted that Gabaculine has the followingmolecular formula: C₇H₉NO₂, and its molecular weight is 139.152 g/mol.

According to another preferred embodiment, the inhibitor may beGabaculine analogue 8 (LHJ-II-79). It should be appreciated that thisanalogue has the following chemical name:1-carboxy-3-amino-4-bis(trifluoromethyl)vinylidene cyclopentanehydrochloride and has the following molecular formula: C₉O₂N₁Cl₁F₆H₈.

It should be noted that any derivatives and stereoisomers of theseinhibitors are also contemplated within the scoop of the invention. Theterm “stereoisomer” is a general term for all isomers of individualmolecules that differ only in the orientation of their atoms in space.It includes mirror image isomers (enantiomers), geometric (cis/trans)isomers, and isomers of compound with more than one chiral center thatare not mirror images of one another (diastereoisomers).

According to an alternative embodiment, inhibition of OAT may beachieved by inhibition of the expression of this enzyme. The expressionof ornithine aminotransferase (OAT) may be inhibited by administering tothe treated subject an inhibitory effective amount of a nucleic acidmolecule comprising at least one target specific sequence, whichsequence is complementary to a target ribonucleotide sequence comprisedwithin OAT mRNA.

It should be appreciated that according to a specific embodiment the OATis the human OAT as referred to by the mRNA sequence of GenBankAccession No. NM_(—)000274, as encoded by genomic region 166400-187979of human chromosome 10, referred to in GenBank Accession No. NT 035040,incorporated herein by reference.

More specifically, a nucleic acid molecule may be selected from thegroup consisting of an antisense DNA or RNA molecule, a ribonucleic acidmolecule having endonuclease activity (ribozyme) and a small interferingRNA (siRNA) specific for said OAT.

The term “nucleic acid” refers to a polymer of nucleotides, or apolynucleotide, as described above. The term is used to designate asingle molecule, or a collection of molecules. Nucleic acids may besingle stranded or double stranded, and may include coding regions andregions of various control elements, as described below.

According to one embodiment, inhibition of OAT expression may beperformed using an anti-sense technology. By “antisense nucleic acid” itis meant a non-enzymatic nucleic acid molecule that binds to target RNAby means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid)interactions and alters the activity of the target RNA. Typically,antisense molecules will be complementary to a target sequence along asingle contiguous sequence of the antisense molecule. However, incertain embodiments, an antisense molecule may bind to substrate suchthat the substrate molecule forms a loop, and/or an antisense moleculemay bind such that the antisense molecule forms a loop. Thus, theantisense molecule may be complementary to two (or even more)non-contiguous substrate sequences or two (or even more) non-contiguoussequence portions of an antisense molecule may be complementary to atarget sequence or both.

By “complementarity” is meant that a nucleic acid can form hydrogenbond(s) with another nucleic acid sequence by either traditionalWatson-Crick or other non-traditional types. In reference to the nucleicmolecules used by the present invention, the binding free energy for anucleic acid molecule with its target or complementary sequence issufficient to allow the relevant function of the nucleic acid toproceed, e.g., ribozyme cleavage, recruitment of RICA by siRNA.

Inhibition of OAT expression may be performed according to anotherspecific embodiment, using ribozyme specific for OAT. Ribozymes are RNAmolecules having an enzymatic activity which is able to repeatedlycleave other separate RNA molecules in a nucleotide base sequencespecific manner. It is said that such enzymatic RNA molecules can betargeted to virtually any RNA transcript and efficient cleavage has beenachieved in vitro.

Six basic varieties of naturally-occurring enzymatic RNAs are knownpresently. Each can catalyze the hydrolysis of RNA phosphodiester bondsin-trans (and thus can cleave other RNA molecules) under physiologicalconditions. In general, enzymatic nucleic acids act by first binding toa target RNA. Such binding occurs through the target binding portion ofan enzymatic nucleic acid which is held in close proximity to anenzymatic portion of the molecule that acts to cleave the target RNA.Thus, the enzymatic nucleic acid first recognizes and then binds atarget RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After an enzymatic nucleic acid hasbound and cleaved its RNA target, it is released from that RNA to searchfor another target and can repeatedly bind and cleave new targets.

In yet another specific embodiment, siRNA may be used by the method ofthe invention for inhibiting the expression of OAT. The term “siRNAs”refers to short interfering RNAs. The term “RNA interference” or “RNAi”refers to the silencing or decreasing of gene expression by siRNAs. Itis the process of sequence-specific, post-transcriptionalsequence-specific gene silencing in animals and plants, initiated bysiRNA that is homologous in its duplex region to the sequence of thesilenced gene. The gene may be endogenous or exogenous to the organism,integrated into a chromosome or present in a transfection vector whichis not integrated into the genome. The expression of the gene is eithercompletely or partially inhibited. RNAi may also inhibit the function ofa target RNA, and said function may be completely or partiallyinhibited.

RNAi is a multistep process. In a first step there is cleavage of largedsRNAs, through the action of the Dicer enzyme (an RNase IIIendonuclease), into 21-23 ribonucleotides-long double stranded effectormolecules called small interfering RNAs (siRNAs). These siRNAs duplexesthen associate with an endonuclease-containing complex, known asRNA-induced silencing complex (RISC). The RISC specifically recognizesand cleaves the endogenous mRNAs containing a sequence complementary toone of the siRNA strands. One of the strands of the double-strandedsiRNA molecule comprises a nucleotide sequence that is complementary toa nucleotide sequence of the endogenous mammalian target gene,specifically OAT or a portion thereof, and the second strand of thedouble-stranded siRNA molecule comprises a nucleotide sequencesubstantially similar to the nucleotide sequence of the endogenousmammalian target gene (OAT) or a portion thereof.

In some embodiments, siRNAs comprise a duplex, or double-strandedregion, of about 18-25 nucleotides long. Often siRNAs contain from abouttwo to four unpaired nucleotides at the 3′ end of each strand. At leasta portion of one strand of the duplex or double-stranded region of asiRNA is substantially homologous to or substantially complementary to atarget sequence within OAT RNA molecule. The strand complementary to atarget RNA molecule is the “antisense strand”, the strand homologous tothe target RNA molecule is the “sense strand” (which is alsocomplementary to the siRNA antisense strand). siRNAs may also containadditional sequences. Non-limiting examples of such sequences includelinking sequences, or loops, as well as stem and other foldedstructures. siRNAs appear to function as key intermediaries intriggering RNA interference in invertebrates and in vertebrates, and intriggering sequence-specific RNA degradation during posttranscriptionalgene silencing.

It should be appreciated that inhibition of OAT expression or activitymay be achieved by a combination therapy combining any of the expressionor activity inhibitors described above.

The term “combination therapy” can mean concurrent or consecutiveadministration of two or more agents. For example, concurrentadministration can mean one dosage form in which the two or more agentsare contained whereas consecutive administration can mean separatedosage forms administered to the patient at different times and maybeeven by different routes of administration.

In yet another embodiment, the method of the invention is intended forthe treatment of pathologic proliferative disorder.

As used herein, the term “disorder” refers to a condition in which thereis a disturbance of normal functioning. A “disease” is any abnormalcondition of the body or mind that causes discomfort, dysfunction, ordistress to the person affected or those in contact with the person.Sometimes the term is used broadly to include injuries, disabilities,syndromes, symptoms, deviant behaviors, and atypical variations ofstructure and function, while in other contexts these may be considereddistinguishable categories. It should be noted that the terms “disease”,“disorder”, “condition” and “illness”, are equally used herein.

According to a preferred embodiment, the method of the invention isspecifically applicable for the treatment of malignant proliferativedisorders. As used herein to describe the present invention, “cancer”,“tumor” and “malignancy” all relate equivalently to a hyperplasia of atissue or organ. If the tissue is a part of the lymphatic or immunesystems, malignant cells may include non-solid tumors of circulatingcells. Malignancies of other tissues or organs may produce solid tumors.

In general, the methods and compositions of the present invention may beused in the treatment of non-solid and solid tumors.

Malignancy, as contemplated in the present invention may be selectedfrom the group consisting of melanomas, carcinomas, leukemias, lymphomasand sarcomas. Malignancies that may find utility in the presentinvention can comprise but are not limited to hematological malignancies(including leukemia, lymphoma and myeloproliferative disorders),hypoplastic and aplastic anemia (both virally induced and idiopathic),myelodysplastic syndromes, all types of paraneoplastic syndromes (bothimmune mediated and idiopathic) and solid tumors (including bladder,rectum, stomach, cervix, ovarian, renal, lung, liver, breast, colon,prostate GI tract, pancreas and Karposi). More particularly, accordingto a preferred embodiment, the OAT inhibitors used by the method of theinvention or any composition comprising the same according to theinvention, can be used for the treatment or inhibition of non-solidcancers, e.g. hematopoietic malignancies such as all types of leukemia,e.g. acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML),chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML),myelodysplastic syndrome (MDS), mast cell leukemia, hairy cell leukemia,Hodgkin's disease, non-Hodgkin's lymphomas, Burkitt's lymphoma andmultiple myeloma, as well as for the treatment or inhibition of solidtumors such as tumors in lip and oral cavity, pharynx, larynx, paranasalsinuses, major salivary glands, thyroid gland, esophagus, stomach, smallintestine, colon, colorectum, anal canal, liver, gallbladder,extraliepatic bile ducts, ampulla of Vater, exocrine pancreas, lung,pleural mesothelioma, bone, soft tissue sarcoma, carcinoma and malignantmelanoma of the skin, breast, vulva, vagina, cervix uteri, corpus uteri,ovary, fallopian tube, gestational trophoblastic tumors, penis,prostate, testis, kidney, renal pelvis, ureter, urinary bladder,urethra, carcinoma of the eyelid, carcinoma of the conjunctiva,malignant melanoma of the conjunctiva, malignant melanoma,retinoblastoma, carcinoma of the lacrimal gland, sarcoma of the orbit,brain, spinal cord, vascular system, hemangiosarcoma and Kaposi'ssarcoma.

According to a specifically preferred embodiment, the method of theinvention is particularly applicable for the treatment of hepaotcellularcarcinoma (HCC).

It should be noted that all disorders indicated herein as disorders thatmay be treated by the methods of the invention, may also be treated bythe compositions of the invention described herein after.

According to another embodiment, the different inhibitors of OAT, may beadministered according to the method of the invention in any suitableway. For example, administration comprises oral, intravenous,intraarterial, intramuscular, subcutaneous, intraperitoneal, parenteral,transdermal, intravaginal, intranasal, mucosal, sublingual, topical,rectal or subcutaneous administration, or any combination thereof.

According to another embodiment, the treated subject may be a mammaliansubject. Although the methods of the invention are particularly intendedfor the treatment of proliferative disorders in humans, other mammalsare included. By way of non-limiting examples, mammalian subjectsinclude monkeys, equines, cattle, canines, felines, mice, rats and pigs.

The terms “treat, treating, treatment” as used herein and in the claimsmean ameliorating one or more clinical indicia of disease activity in apatient having a pathologic disorder.

“Treatment” refers to therapeutic treatment. Those in need of treatmentare mammalian subjects suffering from any pathologic disorder. By“patient” or “subject in need” is meant any mammal for whichadministration of the OAT inhibitors, or any pharmaceutical compositionof the invention is desired, in order to prevent, overcome or slow downsuch infliction.

To provide a “preventive treatment” or “prophylactic treatment” isacting in a protective manner, to defend against or prevent something,especially a condition or disease.

The terms “effective amount” or “sufficient amount” mean an amountnecessary to achieve a selected result. The “effective treatment amount”is determined by the severity of the disease in conjunction with thepreventive or therapeutic objectives, the route of administration andthe patient's general condition (age, sex, weight and otherconsiderations known to the attending physician).

As shown by Example 3, in vivo injection of 500 μg/kg of body weight ofthe Gabaculine inhibitor significantly suppressed tumor growth. Based onthese results, a daily amount of such preferred inhibitor may containbetween about 0.01 to 5000, preferably, 0.5 to 50 mg per kg of bodyweight of the Gabaculine inhibitor.

Therapeutic formulations may be administered in any conventional dosageformulation. Formulations typically comprise at least one activeingredient, as defined above, together with one or more acceptablecarriers thereof.

Each carrier should be both pharmaceutically and physiologicallyacceptable in the sense of being compatible with the other ingredientsand not injurious to the patient. Formulations include those suitablefor oral, rectal, nasal, or parenteral (including subcutaneous,intramuscular, intravenous and intradermal) administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods well known in the art of pharmacy. Thenature, availability and sources, and the administration of all suchcompounds including the effective amounts necessary to produce desirableeffects in a subject are well known in the art and need not be furtherdescribed herein.

In a second aspect, the invention relates to a pharmaceuticalcomposition for the treatment of a pathologic proliferative disorder.The composition of the invention comprises as an active ingredient anornithine aminotransferase (OAT) inhibitor, that may be capable ofinhibiting the expression or the enzymatic activity of said OAT.

According to one specific embodiment, the composition of the inventionmay comprise as an active ingredient an ornithine aminotransferase (OAT)inhibitor selected from the group consisting of5-amino-1,3-hexadienyl-carboxylic acid (Gabaculine), Gabaculine analogue8 (LHJ-II-79), 5-fluoromethylornithine (6-fluoro-2,5-diaminohexanoicacid, 5-FMOrn) and (S)-2-amino-4-amino-oxybutyric acid (Canaline).

According to a specifically preferred embodiment, the composition of theinvention comprises as an active ingredient the OAT inhibitor,5-amino-1,3-hexadienyl-carboxylic acid (Gabaculine).

According to another specifically preferred embodiment, the compositionof the invention comprises as an active ingredient the OAT inhibitor,Gabaculine analogue 8 (LHJ-II-79).

In an alternative embodiment, the composition of the invention comprisesas an active ingredient an OAT inhibitor that may be a nucleic acidmolecule comprising at least one target specific sequence, whichsequence is complementary to a target ribonucleotide sequence comprisedwithin OAT mRNA. More specifically, such nucleic acid molecule may beselected from the group consisting of an antisense DNA or RNA molecule,a ribonucleic acid molecule having endonuclease activity (ribozyme) anda small interfering RNA (siRNA) specific for said OAT.

According to another preferred embodiment, the composition of theinvention is intended for the treatment of a pathologic proliferativedisorder, preferably, a malignant proliferative disorder of any one ofsolid and non-solid tumor selected from the group consisting ofcarcinoma, sarcoma, melanoma, lymphoma and leukemia. More specifically,the composition of the invention is particularly applicable for thetreatment of carcinoma such as liver, breast, bladder, rectum, stomach,cervix, ovarian, colon, renal or prostate carcinoma.

According to a specifically preferred embodiment, the composition of theinvention is intended for the treatment of hepatocellular carcinoma(HCC).

The pharmaceutical compositions of the invention generally comprise abuffering agent, an agent which adjusts the osmolarity thereof, andoptionally, one or more pharmaceutically acceptable carriers, excipientsand/or additives as known in the art. Supplementary active ingredientscan also be incorporated into the compositions. The carrier can besolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, and vegetable oils.The proper fluidity can be maintained, for example, by the use of acoating, such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants.

As used herein “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents and the like. The use of such media and agents for pharmaceuticalactive substances is well known in the art. Except as any conventionalmedia or agent is incompatible with the active ingredient, its use inthe therapeutic composition is contemplated.

The invention further relates to an ornithine aminotransferase (OAT)inhibitor for use in the treatment of pathologic proliferative disordersin a subject in need thereof.

In a further aspect, the invention provides for the use of an ornithineaminotransferase (OAT) inhibitor for the preparation of a pharmaceuticalcomposition for the treatment of a pathologic proliferative disorder ina subject in need thereof.

According to one embodiment, the ornithine aminotransferase (OAT)inhibitor used by the invention may be capable of inhibiting OATenzymatic activity, such inhibitor may be selected from the groupconsisting of 5-amino-1,3-hexadienyl-carboxylic acid (Gabaculine),Gabaculine analogue 8 (LHJ-II-79), 5-fluoromethylornithine(6-fluoro-2,5-diaminohexanoic acid, 5-FMOrn) and(S)-2-amino-4-amino-oxybutyric acid (Canaline). According to aspecifically preferred embodiment, the inhibitor may be5-amino-1,3-hexadienyl-carboxylic acid (Gabaculine). According toanother specifically preferred embodiment, the inhibitor may beGabaculine analogue 8 (LHJ-II-79).

In yet another alternative embodiment, the invention provides the use ofa nucleic acid molecule comprising at least one target specificsequence, which sequence is complementary to a target ribonucleotidesequence comprised within OAT mRNA, as an OAT inhibitor.

More specifically, the nucleic acid molecule may be a ribonucleic acidmolecule selected from the group consisting of an antisense DNA or RNAmolecule, a ribonucleic acid molecule having endonuclease activity(ribozyme) and a small interfering RNA (siRNA) specific for said OAT.

According to another embodiment, the invention relates to the use ofdifferent OAT inhibitors for the preparation of composition for thetreatment of a malignant pathologic proliferative disorder such as solidand non-solid tumor selected from the group consisting of carcinoma,sarcoma, melanoma lymphoma and leukemia.

More particularly, such carcinoma may be a liver, breast, bladder,rectum, stomach, cervix, ovarian, colon, renal or prostate carcinoma.

According to a specifically preferred embodiment, the invention relatesto the use of different OAT inhibitors for the preparation ofcomposition for the treatment of hepatocellular carcinoma (HCC).

In a fourth aspect, the invention relates to a diagnostic method for thediagnosis of a pathologic proliferative disorder. The diagnostic methodof the invention comprises the steps of (a) determining the level of OATexpressed in a biological sample obtained from said subject; (b)determining the level of one or more control genes expressed in saidbiological sample obtained from said subject; and (c) comparing thelevel of expression of OAT in said sample according to step a) and thelevel of expression of each of said one or more control genes in saidsample according to step b) with the level of OAT and the level of oneor more control reference genes in a control sample. It should be notedthat detecting differential expression of OAT in the comparison of stepc) is indicative of that said subject is suffering of said proliferativedisorder.

It should be noted that the diagnostic method of the invention is basedon the finding that OAT is over expressed in samples of HCC. Thisdifferential expression is the basis for the development of thediagnostic method of the invention. “Differentially expressed” can alsoinclude a measurement of the RNA or protein encoded by the OAT gene ofthe invention in a sample or population of samples as compared with theamount or level of RNA or protein expression in a second sample orpopulation of samples. Differential expression can be determined asdescribed herein and as would be understood by a person skilled in theart. The term “differentially expressed” or “changes in the level ofexpression” refers to an increase or decrease in the measurableexpression level of the OAT gene as measured by the amount of RNA and/orthe amount of protein in a sample as compared with the measurableexpression level of the OAT gene in a second sample. The term“differentially expressed” or “changes in the level of expression” canalso refer to an increase or decrease in the measurable expression levelof the OAT gene in a population of samples as compared with themeasurable expression level of the OAT gene in a second population ofsamples. As used herein, “differentially expressed” can be measuredusing the ratio of the level of expression of the OAT gene as comparedwith the mean expression level of the OAT gene of a control wherein theratio is not equal to 1.0. Differentially expressed can also be measuredusing p-value. When using p-value, the OAT gene is identified as beingdifferentially expressed as between a first and second population whenthe p-value is less than 0.1. More preferably the p-value is less than0.05. Even more preferably the p-value is less than 0.01. Morepreferably still the p-value is less than 0.005. Most preferably thep-value is less than 0.001. When determining differentially expressionon the basis of the ratio, an RNA or protein is differentially expressedif the ratio of the level of expression in a first sample as comparedwith a second sample is greater than or less than 1.0. For example, aratio of greater than 1.2, 1.5, 1.7, 2, 3, 4, 10, 20 or a ratio lessthan 1, for example 0.8, 0.6, 0.4, 0.2, 0.1. 0.05. In another embodimentof the invention a nucleic acid transcript or is differentiallyexpressed if the ratio of the mean of the level of expression of a firstpopulation as compared with the mean level of expression of the secondpopulation is greater than or less than 1.0 For example, a ratio ofgreater than 1.2, 1.5, 1.7, 2, 3, 4, 10, 20 or a ratio less than 1, forexample 0.8, 0.6, 0.4, 0.2, 0.1. 0.05. In another embodiment of theinvention a nucleic acid transcript or is differentially expressed ifthe ratio of its level of expression in a first sample as compared withthe mean of the second population is greater than or less than 1.0 andincludes for example, a ratio of greater than 1.2, 1.5, 1.7, 2, 3, 4,10, 20, or a ratio less than 1, for example 0.8, 0.6, 0.4, 0.2, 0.1.0.05.

More specifically, “Differentially increased expression” or “upregulation” refers to genes, such as the OAT gene, which demonstrate atleast 10% or more, for example, 20%, 30%, 40%, or 50%, 60%, 70%, 80%,90% or more or 1.1 fold, 1.2 fold, 1.4 fold, 1.6 fold, 1.8 fold, or moreincrease in gene expression (as measured by RNA expression or proteinexpression), relative to a control.

The term “overexpression” refers to the production of a gene product inan organism or a certain tissue that exceeds levels of production innormal organisms or tissues. Example 1, clearly demonstrateoverexpression of OAT in HCC cells compared to normal cells.

“Differentially decreased expression” or “down regulation” refers togenes which demonstrate at least 10% or more, for example, 20%, 30%,40%, or 50%, 60%, 70%, 80%, 90% or a less than 1.0 fold, 0.8 fold, 0.6fold, 0.4 fold, 0.2 fold, 0.1 fold or less decrease in gene expression(as measured by RNA expression or protein expression), relative to acontrol.

A gene expression pattern of the OAT gene can result from themeasurement of expression of the RNA or protein products of the OAT geneof the invention and can be done using any known technique. For exampletechniques to measure expression of the RNA products of the OAT gene ofthe invention includes, PCR based methods (including RT-PCR) and non PCRbased method as well as micro-array analysis. To measure proteinproducts of the OAT gene of the invention, techniques include westernblotting and ELISA analysis.

More particularly, according to one embodiment, determination of theexpression of the OAT gene according to step (a) and determination ofthe expression of one or more control reference genes according to step(b) of the method of the invention, may be performed by a methodcomprising the step of contacting said sample or any nucleic acid oramino acid product obtained therefrom with at least one detectingmolecule or a collection of at least two detecting molecules specificfor determination of the expression of the OAT gene and of one or morecontrol reference genes.

It should be appreciated that the detection step may be performed usingthe tested sample as obtained from the tested subject, or alternatively,may be performed using any constituent or material derived or preparedtherefrom. As a non-limiting example, it should be noted that the methodof the invention further encompasses the use of nucleic acid moleculesand or proteins prepared from the tested sample.

According to one preferred embodiment the detecting molecule used forthe diagnostic method of the invention may be an isolated nucleic acidmolecule or an isolated amino acid molecule, or any combination thereof.

According to one alternative and preferred embodiment, the method of theinvention uses as a detecting molecule an isolated nucleic acidmolecule. More specifically, such nucleic acid molecule may be anisolated oligonucleotide which specifically hybridizes to a nucleic acidsequence of the RNA products of OAT.

Accordingly, the expression of the OAT gene and of the control referencegene may be determined according to a preferred embodiment, using anucleic acid amplification assay such as PCR, Real Time PCR, microarrays, in situ Hybridization and Comparative Genomic Hybridization.

According to an alternative embodiment, the method of the invention usesan isolated amino acid molecule as the detecting molecule. Suchdetecting molecule may be therefore an isolated polypeptide which bindsselectively to the protein product of OAT.

Accordingly, the detecting molecule for the control reference genes maybe an isolated polypeptide which binds selectively to a protein productof at least one control reference gene. Such control genes may be forexample, HSPCB, RPS9, RPL32 or β-actin.

According to a specifically preferred embodiment, the detecting moleculemay be an isolated antibody. In such case, according to anotherembodiment of the invention, the expression may be determined using animmunoassay selected from the group consisting of an ELISA, a RIA, aslot blot, a dot blot, immunohistochemical assay, FACS, a radio-imagingassay or a Western blot.

It should be appreciated that in case the detecting molecule may be ananti-OAT antibody, the antibody used for of the invention may be amonoclonal or polyclonal antibody.

It should be noted that the term “antibody” is meant to include intactmolecules as well as fragments thereof, such as, for example, Fab andF(ab′)2, which are capable of binding antigen.

It will be appreciated that Fab and F(ab′)₂ and other fragments of theantibodies are within the scope of the present invention and may be usedfor the kits and the diagnostic methods disclosed herein for intactantibody molecules. Such fragments are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments).

In a further embodiment, the diagnostic method of the invention isintended for the diagnosis and monitoring of a pathological disorder ina mammalian subject.

According to one embodiment of the invention the biological sample ismalignant, e.g., it is a solid tumor or hematopoietic tumor sample. Thesolid tumor can, for example, be of the types: carcinoma, glyoma,adenocarcinoma, squameous cell carcinoma, teratocarcinoma, mesotheliomaor melanoma. The hematopoietic tumor can, for example, be lymphoma orleukemia. In some embodiments of the present invention the solid tumoris a primary tumor, or a metastasis thereof, and it originates from anorgan such as, for example, liver, prostate, bladder, breast, ovary,cervix, colon, skin, intestine, stomach, uterus (including embryo) andpancreas. These conditions include myeloma, breast carcinoma andmetastatic breast carcinoma.

The present invention relates, in some embodiments, to diagnosticassays, which in some embodiments, utilizes a biological sample takenfrom a subject (patient), which for example may comprise any biologicalsample, such as body fluid or secretion including but not limited toseminal plasma, blood, serum, urine, prostatic fluid, seminal fluid,semen, the external secretions of the skin, respiratory, intestinal, andgenitourinary tracts, tears, cerebrospinal fluid, sputum, saliva, milk,peritoneal fluid, pleural fluid, cyst fluid, secretions of the breastductal system (and/or lavage thereof), broncho alveolar lavage, lavageof the reproductive system, lavage of any other part of the body orsystem in the body, samples of any organ including but not limited tolung, colon, ovarian and/or breast tissue, feaces or a tissue sample,any cells derived therefrom, or any combination thereof. In someembodiments, the term encompasses samples of in vitro or ex vivo cellculture or cell culture constituents. The sample can optionally bediluted with a suitable eluant before contacting the sample with thedetecting molecule/s of the invention and/or performing any otherdiagnostic assay.

As used herein, “patient”, “subject” or “individual” refers to a mammal,preferably human, who is diagnosed by the method of the invention.

As used herein, the term “control” or “control sample” includes positiveor negative controls. In the context of this invention the term“positive control” refers to one or more samples isolated from anindividual or group of individuals who are classified as suffering of apathologic proliferative disorder. The term “negative control” refers toone or more samples isolated from an individual or group of individualswho are healthy subjects.

The invention further provides a diagnostic kit for non-invasivedetection and monitoring of a pathologic disorder in a mammaliansubject. The kit of the invention may comprise: (a) means for obtaininga sample of said subject; (b) at least one detecting molecule or acollection of at least two detecting molecules specific fordetermination of the expression of OAT.

It should be noted that the kit of the invention may optionallycomprise, (c) at least one detecting molecule or a collection of atleast two detecting molecules specific for determination of theexpression of at least one control reference gene or a collection of atleast two control reference genes. Such control reference genes may beHSPCB, RPS9, RPL32 or β-actin. The kit of the invention furthercomprises (d) at least one control sample selected from at least one ofa negative control sample and a positive control sample; (e)instructions for carrying out the detection and quantification ofexpression of said OAT and of at least one control reference gene insaid sample; and (f) instructions for evaluating the differentialexpression of said OAT in said sample and optionally of a controlreference gene in said sample as compared to the expression of said OATand optionally control reference gene in said control sample.

According to one embodiment, the detecting molecule comprised within thekit of the invention may be an isolated nucleic acid molecule or anisolated amino acid molecule, or any combination thereof.

According to one specific and preferred embodiment, the detectingmolecule comprised within the kit of the invention may be an isolatednucleic acid molecule. Such molecule may be preferably, an isolatedoligonucleotide which specifically hybridizes to a nucleic acid sequenceof the RNA products of OAT.

According to a preferred embodiment, such oligonucleotide may be a pairof primer or nucleotide probe or any combination, mixture or collectionthereof.

According to another preferred optional embodiment, the kit of theinvention may further comprise at least one reagent for performing anucleic acid amplification based assay. Such nucleic acid amplificationassay may be any one of PCR, Real Time PCR, micro arrays, in situHybridization and Comparative Genomic Hybridization.

According to a specifically preferred embodiment such detecting moleculemay be an isolated antibody.

It should be noted that the kit of the invention may optionally furthercomprises at least one reagent for performing an immuno assay, such asELISA, a RIA, a slot blot, a dot blot, immunohistochemical assay, FACS,a radio-imaging assay, Western blot or any combination thereof.

According to a preferred embodiment, the kits provided by the inventionmay further comprise suitable means and reagents for preparing orisolating at least one of nucleic acids and amino acids from theexamined sample.

In another embodiment, the present invention relates in part to kitscomprising sufficient materials for performing one or more methodsdescribed herein. In preferred embodiments, a kit includes one or morematerials selected from the following group in an amount sufficient toperform at least one assay.

Thus, according to another preferred optional embodiment, the kit of theinvention may further comprise at least one reagent for performing anucleic acid amplification based assay. Such nucleic acid amplificationassay may be any one of PCR, Real Time PCR, micro arrays, in situHybridization and Comparative Genomic Hybridization.

Control nucleic acid members may be present on the array includingnucleic acid members comprising oligonucleotides or nucleic acidscorresponding to genomic DNA, housekeeping genes, vector sequences,plant nucleic acid sequence, negative and positive control genes, andthe like. Control nucleic acid members are calibrating or control geneswhose function is not to tell whether a particular “key’ gene ofinterest is expressed, but rather to provide other useful information,such as background or basal level of expression. Such control samplesmay be for example, HSPCB, RPS9, RPL32 and β-actin. Optionally, othercontrol nucleic acids may be spotted on the array and used as targetexpression control nucleic acids.

According to an alternative embodiment, the detecting molecule comprisedwithin the kit of the invention may be an isolated amino acid molecule,for example, an isolated polypeptide which binds selectively to theprotein product of OAT

It should be noted that the kit of the invention may therefore furthercomprises at least one reagent for performing an immuno assay, such asELISA, a RIA, a slot blot, a dot blot, immunohistochemical assay, FACS,a radio-imaging assay, Western blot or any combination thereof.

According to a preferred embodiment, the kits provided by the inventionmay further comprise suitable means and reagents for preparing orisolating at least one of nucleic acids and amino acids from saidsample.

For RT-PCR kits, the kits generally comprise pre-selected primersspecific for particular RNA products (e.g., an exon(s), an intron(s), anexon junction(s), and an exon-intron junction(s)) of the OAT gene of theinvention. The RT-PCR kits may also comprise enzymes suitable forreverse transcribing and/or amplifying nucleic acids (e.g., polymerasessuch as Taq), and deoxynucleotides and buffers needed for the reactionmixture for reverse transcription and amplification. The RT-PCR kits mayalso comprise probes specific for RNA products of the OAT gene of theinvention. The probes may or may not be labeled with a detectable label(e.g., a fluorescent label). Each component of the RT-PCR kit isgenerally in its own suitable container. Thus, these kits generallycomprise distinct containers suitable for each individual reagent,enzyme, primer and probe. Further, the RT-PCR kits may compriseinstructions for performing the assay and methods for interpreting andanalyzing the data resulting from the performance of the assay.

For antibody based kits, the kit can comprise, for example: (1) a firstantibody (which may or may not be attached to a support) which binds toprotein of interest (e.g., a protein product of the OAT gene of theinvention); and, optionally, (2) a second, different antibody whichbinds to either the protein, or the first antibody and is conjugated toa detectable label (e.g., a fluorescent label, radioactive isotope orenzyme).

The antibody-based kits may also comprise beads for conducting animmunoprecipitation.

Each component of the antibody-based kits is generally in its ownsuitable container. Thus, these kits generally comprise distinctcontainers suitable for each antibody. Further, the antibody-based kitsmay comprise instructions for performing the assay and methods forinterpreting and analyzing the data resulting from the performance ofthe assay.

It should be thus appreciated that any of the kits of the invention mayoptionally further comprises solid support, such as plates, beads, tubeor containers. These may be specifically adopted for performingdifferent detection steps or any nucleic acid amplification based assayor immuno assay, as described for example by the method of theinvention. It should be further noted that any substance or ingredientcomprised within any of the kits of the invention may be attached,embedded, connected or linked to any solid support.

It should be noted that any of the detecting molecules used by thecompositions, methods and kits of the invention may be labeled by adetectable label. The term “detectable label” as used herein refers to acomposition or moiety that is detectable by spectroscopic,photochemical, biochemical, immunochemical, electromagnetic,radiochemical, or chemical means such as fluorescence, chemifluoresence,or chemiluminescence, or any other appropriate means. Preferreddetectable labels are fluorescent dye molecules, or fluorochromes, suchfluorescein, phycoerythrin, CY3, CY5, allophycocyanine, Texas Red,peridenin chlorophyll, cyanine, FAM, JOE, TAMRA, tandem conjugates suchas phycoerythrin-CY5, and the like. These examples are not meant to belimiting.

It is to be understood that any polynucleotide or polypeptide or anycombination thereof described by the invention may be useful as a markerfor a disease, disorder or condition, and such use is to be considered apart of this invention.

It should be appreciated that all method and kits described herein,preferably comprises any of the compositions of the invention.

It should be recognized that the nucleic acid sequences and/or aminoacid sequences used by the kits of the present invention relate, in someembodiments, to their isolated form, as isolated polynucleotides(including for all transcripts), oligonucleotides (including for allsegments, amplicons and primers), peptides (including for all tails,bridges, insertions or heads, optionally including other antibodyepitopes as described herein) and/or polypeptides (including for allproteins). It should be noted that the terms “oligonucleotide” and“polynucleotide”, or “peptide” and “polypeptide”, may optionally be usedinterchangeably.

Disclosed and described, it is to be understood that this invention isnot limited to the particular examples, methods steps, and compositionsdisclosed herein as such methods steps and compositions may varysomewhat. It is also to be understood that the terminology used hereinis used for the purpose of describing particular embodiments only andnot intended to be limiting since the scope of the present inventionwill be limited only by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise.

Throughout this specification and the Examples and claims which follow,unless the context requires otherwise, the word “comprise”, andvariations such as “comprises” and “comprising”, will be understood toimply the inclusion of a stated integer or step or group of integers orsteps but not the exclusion of any other integer or step or group ofintegers or steps. Thus, use of the term “comprising” indicates that thelisted elements are required or mandatory, but that other elements areoptional and may or may not be present. By “consisting of” is meantincluding, and limited to, whatever follows the phrase “consisting of”.Thus, the phrase “consisting of” indicates that the listed elements arerequired or mandatory, and that no other elements may be present. By“consisting essentially of” is meant including any elements listed afterthe phrase, and limited to other elements that do not interfere with orcontribute to the activity or action specified in the disclosure for thelisted elements. Thus, the phrase “consisting essentially of” indicatesthat the listed elements are required or mandatory, but that otherelements are optional and may or may not be present depending uponwhether or not they affect the activity or action of the listedelements.

The following examples are representative of techniques employed by theinventors in carrying out aspects of the present invention. It should beappreciated that while these techniques are exemplary of preferredembodiments for the practice of the invention, those of skill in theart, in light of the present disclosure, will recognize that numerousmodifications can be made without departing from the spirit and intendedscope of the invention.

EXAMPLES Experimental Procedures

Animal Models

-   -   Psammomys obesus (sand rat)—Harlan, Jerusalem, Israel.    -   Athymic Balb/c mice—Harlan, Jerusalem, Israel.        Cell Lines    -   CT-26: is a mouse cell line, provided by ATCC Cat. No. CRL-2638.    -   HUH7: H. is a human cell line described by Nakabyashi et al,        Cancer Research, 42: 3858-3863 (1982).    -   skhep1: is a human cell line, provided by ATCC Cat. No. UTB 52.    -   HepA1-6: a mouse cell line, described by Dalington G. J. et        al., J. Nat Cancer Institute, 64:809-819 (1980).    -   Hep3b-human hepatocellular carcinoma cell line. ATCC™ Cat. No.        HB-8064™.    -   PLC/REF/5: is a human cell line, provided by ATCC™ Cat. No.        CRL-2711.    -   FLC4: is a human cell line, described by Aoki Y. et al, Virogloy        250:140-150 (1998).    -   HepG2: is a human cell line, provided by ATCC Cat. No. HB-8065.    -   LS-180: is a human cell line, provided by ATCC Cat. No. CL-187.        HCC Cell Line Culture Medium

500 ml Mem-Eagle (Earle's), 10% Fetal Calf serum,Penicillin/streptomycin, Sodium bicarbonates 1.5 g/L, Non-essentialamino acids 0.1 Mm and Sodium pyruvate 1 mM.

OAT Inhibitors

-   -   5-amino-1,3-hexadienyl-carboxylic acid (Gabaculine), purchased        from SIGMA-ALDRICH Inc, Saint Louis, Mo., USA, Cat. No. A3539.    -   Gabaculine analogue 8 (LHJ-II-79), which has the following        chemical name:        1-carboxy-3-amino-4-bis(trifluoromethyl)vinylidene cyclopentane        hydrochloride (C₉O₂N₁Cl₁F₆H₈).        DNA Microarray

For performing the DNA microarray analysis, total RNA was extracted fromliver tumors and from normal liver tissue of Psammomys obesus. RNAextraction was repeated from three different tumors and used in threedifferent arrays. Contamination by DNA was eliminated by treatment ofthe RNA with DNAse. Total RNA was used for the synthesis of cDNA. cDNAsof the tumor and the normal tissue were labeled with different dyes andloaded on micro array containing the whole mice genome, and exposuresignals were scanned and quantified. Taking into consideration thevariability attendant upon any biological system, in analyzing the cDNAarrays, the inventors focused only on genes that were differentiallyupregulated or downregulated in all the three liver tumors.

Treatment of HCC Cell Lines with Gabaculine

10⁵ cells of each of the cell lines were grown in 96 wells plates intriplicates. Gabaculine (2000 micro mol/liter) was added for 48 hours.Thymidine incorporation assay and measurement of Alpha Feto-Proteinlevel were performed.

Treatment of HCC Cell Lines with Gabaculine Analogue 8 (LHJ-II-79)

1×10⁵ cells/well of each of Hep3b and HepG2 hepatocellular carcinomacell lines were plated in triplicate and incubated at 37° C. Gabaculineanalogue 8 was added to wells at varying concentrations (0.1 μg/ml, 1μg/ml, 10 μg/ml and 50 μg/ml). Cells returned to incubator for furtherincubation of 48 hours. In the fourth day of the experiment, one ml ofsupernatant from each well was transferred to eppendorf tubes andanalyzed for αFP concentration. αFP levels were checked using the AbbottDiagnostic AxSYM system.

Example 1 Ornithine Aminotransferase (OAT) Overexpression in HCC Tissue

The Psammomys obesus (sand rat) develops spontaneous hepatocellularcarcinoma (HCC), and was therefore used by the present inventors as amodel of proliferative disorder, and more particularly, as a model forhepatocellular carcinoma. The aim of the present study was to identifygenes associated with liver tumorogenesis using the sand rat model andto assess whether specific inhibition of the protein products of suchgenes may suppress HCC growth.

Identification of genes which are overexpressed in hepatocellularcarcinoma tissue was performed using comparative DNA microarray-basedgene expression profiling assay of HCC and normal liver tissue, in aspontaneous HCC-developing sand rat model. Statistical analysis ofmicroarray data revealed seven genes whose expression levels wereincreased by two logs or more in multiple tumors tissues, when comparedto normal liver. One of the most prominent of these genes was ornithineaminotransferase (OAT).

As indicated in the background of the invention, OAT is a mitochondrialenzyme that catalyses the transamination of ornithine to glutamate, andwas found to be a beta-catenin target gene.

Example 2 Inhibition of OAT Leads to Inhibition of HCC Cell LinesProliferation

The significant increase in OAT expression in HCC liver tissue, and thefact that overexpression of OAT enables extracellular glutamineindependent cell growth, which may be advantageous in tumorproliferation, have led the inventors to investigate the potentialeffect of OAT inhibition on HCC proliferation.

5-Amino-1,3-hexadienyl-carboxylic acid (Gabaculine) is known as a potentOAT inhibitor. Gabaculine was next tested in vitro on ten different HCCcell lines. 48 hrs following exposure to Gabaculine, cell proliferation(Table 1) and alpha feto protein (AFP) secretion (Table 2) wereevaluated.

As clearly demonstrated by Table 1, in vitro application of Gabaculineon different HOC cell lines significantly suppressed the proliferation.This effect was clearly shown in three HCC cell lines (Hep3B, PLC andHepA1-6 cell lines, by 46 to 51% p<0.05).

As shown by Table 2, alpha feto protein (AFP) secretion analysis,indicated that Gabaculine significantly decreased AFP secretion by 20%in Hep3B cells (p<0.0005).

TABLE 1 Gabaculine decreases HCC cell proliferation HCC LinesProliferation CPM-B with Gabaculine (20000 umol/L) CT-26 HUH7 skhep1HepA1-6 PLC/REF/5 Line 452 303 289 598 543 Line 398 436 207 739 460 Line391 238 434 Line average 425.00 376.67 244.67   501.50   501.50 Line +Gaba. 491 367 160 220 306 Line + Gaba. 421 360 264 286 315 Line + Gaba.350 312 165 272 244 Line + Gaba. 499 378 287 225 average 440.25 354.25219   250.75     288.33333 tTEST 0.3740275 0.316937 0.278281     0.028344       0.0340366 FLC4 HepG2 LS-180 H3B 2215 Line 47 470 297437 413 Line 48 456 599 521 555 Line 136 475 305 543 Line 264 537 439average 123.75 467.00 434.50   485.00 484 Line + Gaba. 104 526 498 230361 Line + Gaba. 277 512 555 194 466 Line + Gaba. 201 515 528 402 529Line + Gaba. 450 average 194 517.6667 507.75    275.3333 452 tTEST0.1865 0.001358 0.212675      0.032269 0.373485

TABLE 2 Gabaculine significantly decreased AFP secretion in HCC cellsct-26 PLC FLC-4 HEPG2 LS180 HEP3B 2215.00 SKHEP1 HEPAI6 HUH7 line 0.006.03 0.38 5.70 0.00 12.02 1569.40 0.00 0.00 318.46 line 0.03 6.27 0.435.76 0.04 12.06 1486.20 0.00 0.00 243.00 line 5.84 0.36 5.81 0.001520.90 0.00 0.00 291.21 average 0.02 6.05 0.39 5.76 0.01 12.04 1525.500.00 0.00 284.22 line + ga. 0.00 5.99 0.41 5.80 0.00 10.42 1601.20 0.000.02 282.71 line + ga. 0.03 5.85 0.38 5.79 0.00 10.42 1522.00 0.00 0.00234.53 line + ga. 0.00 5.40 0.42 5.29 10.27 1554.80 0.00 0.00 281.06average 0.01 5.75 0.40 5.63 0.00 10.37 1559.33 0.00 0.01 266.10 tTEST0.4044 0.1236 0.3078 0.2613 0.2113 0.0001 0.1837 #DIV/0! 0.2113 0.2721Therefore, in vitro, Gabaculine (20000 umole/litter) treatment,significantly suppressed HCC cell line growth.

Example 3 In Vivo Administration of Gabaculine Significantly SuppressedTumor Growth

The surprising decrease in proliferation of different HCC cell lines, asa result of treatment with OAT inhibitor, encouraged the presentinventors to further explore the possible effect of such inhibition ontumor growth. Therefore, the in vivo effect of OAT inhibition on tumorgrowth was examined using Hep3B HCC-transplanted athymic Balb/c mice.Mice (n=8/group) were injected once with 500 μg/kg of Gabaculinefollowed 48 hrs later by AFP serum levels measurement. PBS was injectedto a matched control group.

As shown by Table 3, in vivo administration of Gabaculine significantlysuppressed tumor growth. Within seven days of treatment, AFP serumlevels decreased by 92% (207,888 to 22,172 pg/ml), in comparison with9.7 fold increase (278,220 to 2,146,927 pg/ml) in treated vs. controls,respectively (p<0.05).

TABLE 3 in-vivo treatment with Gabaculine Group (A) = PBS Day 2 Day 7Day 0 (48 hr. after AFP(3) AFTER AFP(3)/ AFP(1) treatment AFP(2)TREATMENT AFP(2) A-1 2992 347180 9039000 26.03549 A-2 5874 7371006768000 9.181929 A-4 523 45110 58210 1.290401 A-5 1138 721300 536100.074324 A-6 2199 198500 98100 0.494207 A-7 435 105150 263400 2.504993A-9 13 22390 818600 36.56096 A-10 129 49030 76500 1.560269 Average1662.875 278220 2146927.5 9.712822 Group (B + C) = GABACULINE Day 7 Day0 Day 2 AFP(3) AFTER AFP(1) AFP(2) TREATMENT B-1 19920 1004600 122100.012154 B-2 1331 86260 325980 B-5 301 64500 4350 0.067442 B-6 1900158130 96600 0.61089 B-7 3430 667600 32710 0.048996 B-9 833 59920 15000.025033 B-10 989 181110 10950 0.06046 C-2 486 28710 90 0.003135 C-33032 248780 58420 0.234826 C-5 7014 434600 17260 0.039715 C-9 399 4382030 0.000685 C-10 2994 283530 35060 0.123655 Average 2785 207888 221720.080403

In vivo administration of one dose of Gabaculine resulted in suppressionof HCC growth. These results suggest that OAT plays an important role inHCC growth and may serve as a potential therapeutic target.

Example 4

Inhibition of OAT Using Different Concentrations of Gabaculine Analogue8 (LHJ-II-79), Leads to Inhibition of AFP Secretion from HCC Cell Lines

Encouraged by the in vitro and in vivo results of inhibiting OAT usingGabaculine, the inventors next examined the in vitro effect of a furtherOAT inhibitor, Gabaculine analogue 8 (LHJ-II-79), on AFP secretion fromtwo different HCC cell lines. Therefore, Hep3b and HepG2 hepatocellularcarcinoma cell lines were detached from culture dishes usingtrypsin-EDTA, re-plated in triplicates in 12 well culture dishes (1×10⁵cells/well) and incubated at 37° C. Gabaculine analogue 8 was added towells at varying concentrations (0.1 μg/ml, 1 μg/ml, 10 μg/ml and 50μg/ml). After 48 hours, cell supernatant was analyzed for AFPconcentration.

As clearly shown by Table 4, treatment of both CCC cell lines resultedin significant reduction of AFP secretion by the cells. This effect wasconcentration dependent. These results clearly demonstrate thefeasibility of using the Gabaculine analogue 8 (LHJ-II-79), for treatinghepatocellular carcinoma.

TABLE 4 Gabaculine analogue 8 (LHJ-II-79) decreases αFP secretion by HCCcell lines EXP. I AVG results AF ng/ml conc. of HCC cell H3B 1:5 dil,analogue no. analogue line HG2 1:100 dil CONTROL — Hep3b 42.52 8 10ug/ml Hep3b 37.96 8 50 ug/ml Hep3b 18.52 CONTROL — HepG2 34.19 8 10ug/ml HepG2 37.98 8 50 ug/ml HepG2 23.54 Exp. II AVG results AF ng/mlconc. of HCC cell H3B 1:5 dil, analogue no. analogue line HG2 1:100 dilCONTROL — Hep3b 13.42 8 0.1 ug/ml  Hep3b 12.66 8  1 ug/ml Hep3b 11.92 810 ug/ml Hep3b 9.63 8 50 ug/ml Hep3b 9.37 CONTROL — HepG2 11.93 8 0.1ug/ml  HepG2 11.92 8  1 ug/ml HepG2 12.85 8 10 ug/ml HepG2 11.38 8 50ug/ml HepG2 8.54 EXP. III AVG results AF ng/ml conc. of HCC cell H3B 1:5dil, analogue no. analogue line HG2 1:100 dil CONTROL — Hep3b 11.39 8 10ug/ml Hep3b 10.97 8 50 ug/ml Hep3b 6.98 CONTROL — HepG2 12.07 8 10 ug/mlHepG2 10.67 8 50 ug/ml HepG2 7.57 EXP. IV AVG results AF ng/ml conc. ofHCC cell H3B 1:2 dil, analogue no. analogue line HG2 1:50 dil CONTROL —Hep3b 18.45 8 10 ug/ml Hep3b 13.73 8 50 ug/ml Hep3b 11.30 CONTROL —HepG2 13.32 8 10 ug/ml HepG2 12.74 8 50 ug/ml HepG2 8.94

Example 5 In Vivo Administration of Gabaculine Analogue 8 (LHJ-II-79)for Suppression of Tumor Growth

The inventors further explore the possible effect of Gabaculine analogue8 (LHJ-II-79) on tumor growth. Therefore, the in vivo effect of thisinhibitor is examined using Hep3B HCC-transplanted athymic Balb/c mice.Mice (n=8/group) are injected once with 0.1 to 100 μg/kg of Gabaculineanalogue 8 (LHJ-II-79) followed 48 hrs later by AFP serum levels andtumor development (tumor size) measurements. PBS is injected to amatched control group.

1. A method for the treatment of a malignant pathologic proliferativedisorder in a subject in need thereof, comprising the step ofselectively inhibiting the enzymatic activity of ornithineaminotransferase (OAT) by administering to said subject an inhibitoryeffective amount of OAT inhibitor, wherein said inhibitor is any one ofGabaculine analogue 8 (LHJ-II-79) and 5-amino-1,3-hexadienyl-carboxylicacid (Gabaculine) and wherein said malignant pathologic proliferativedisorder is hepatocellular carcinoma (HCC).
 2. The method according toclaim 1, wherein said inhibitor is 5-amino-1,3-hexadienyl-carboxylicacid (Gabaculine).
 3. The method according to claim 1, wherein saidinhibitor is Gabaculine analogue 8 (LHJ-II-79).
 4. The method accordingto claim 1, wherein inhibiting the expression of ornithineaminotransferase (OAT) is performed by administering to said subject aninhibitory effective amount of a nucleic acid molecule comprising atleast one target specific sequence, which sequence is complementary to atarget ribonucleotide sequence comprised within OAT mRNA and whereinsaid nucleic acid molecule is selected from the group consisting of anantisense DNA or RNA molecule, a ribonucleic acid molecule havingendonuclease activity (ribozyme) and a small interfering RNA (siRNA)specific for said OAT.
 5. A method for the treatment of hepatocellularcarcinoma (HCC) in a subject in need thereof, comprising the step ofadministering to said subject a therapeutically effective amount ofGabaculine analogue 8 (LHJ-II-79).
 6. A method for the treatment ofhepatocellular carcinoma (HCC) in a subject in need thereof, comprisingthe step of administering to said subject a therapeutically effectiveamount of 5-amino-1,3-hexadienyl-carboxylic acid (Gabaculine).